PROJECT SUMMARY Tumors are complex ecosystems containing cells with different functions, which interact to maintain the cancerous environment. Profiling tumors has been severely limited by their cellular diversity, because the methods used for molecular analysis of whole tumor samples might not reflect any single tumor cell accurately. Measuring the RNA composition of 10,000s of individual cells from a tumor could turn this heterogeneity into a benefit, by revealing druggable vulnerabilities in immune and stromal cells, as well as potentially dangerous tumor-initiating transcriptomes in rare cancer cells. To address this opportunity, we propose to adapt a powerful microfluidic platform that we have developed, known as inDrops, for low-cost high-throughput single- cell profiling in cancer biology and specifically for biopsy samples. Relatively little additional technology development is required to make this method practical for analysis of individual tumors for basic research or in a clinical setting. This project focuses on solidifying the advantages of this transformative novel platform through achievable steps. Currently, inDrops is the only single-cell transcriptomics platform with the scale and cell recovery rate required to make it a practical tool for biopsy analysis. Our specific aims focus on demonstrating the method on tumor samples, optimizing microfluidic engineering for robust work with biopsies, adapting the method to work with pre-fixed cells, adapting the method for screening with targeted gene panels, and integrating the resulting data with histological analysis so as to maximally facilitate diagnostically and prognostically relevant discoveries. Together, these advances will transform inDrops into a powerful method for wide use in cancer biology.